TAILIEUCHUNG - Báo cáo khoa học: Effects on protease inhibition by modifying of helicase residues in hepatitis C virus nonstructural protein 3

This study of the full-length bifunctional nonstructural protein 3 from hep-atitis C virus (HCV) has revealed that residues in the helicase domain affect the inhibition of the protease. Two residues (Q526 and H528), appar-ently located in the interface between the S2 and S4 binding pockets of the substrate binding site of the protease, were selected for modification, and three enzyme variants (Q526A, H528A and H528S) were expressed, puri-fied and characterized. | ỊFEBS Journal Effects on protease inhibition by modifying of helicase residues in hepatitis C virus nonstructural protein 3 Goran Dahl1 Anja Sandstrom2 Eva Akerblom2 and U. Helena Danielson1 1 Department of Biochemistry and Organic Chemistry Uppsala University Sweden 2 Department of MedicinalChemistry Organic PharmaceuticalChemistry Uppsala University Sweden Keywords full length hepatitis C virus inhibition nonstructuralprotein 3 protease Correspondence U. H. Danielson Department of Biochemistry and Organic Chemistry Uppsala University BMC Box576 SE-751 23 Uppsala Sweden Fax 46 18 558431 Tel 46 18 4714545 E-mail Received 4 May 2007 revised 17 August 2007 accepted 25 September 2007 doi This study of the full-length bifunctional nonstructural protein 3 from hepatitis C virus HCV has revealed that residues in the helicase domain affect the inhibition of the protease. Two residues Q526 and H528 apparently located in the interface between the S2 and S4 binding pockets of the substrate binding site of the protease were selected for modification and three enzyme variants Q526A H528A and H528S were expressed purified and characterized. The substitutions resulted in indistinguishable Km values and slightly lower kcat values compared to the wild-type. The Ki values for a series of structurally diverse protease inhibitors were affected by the substitutions with increases or decreases up to 10-fold. The inhibition profiles for H528A and H528S were different confirming that not only did the removal of the imidazole side chain have an effect but also that minor differences in the nature of the introduced side chain influenced the characteristics of the enzyme. These results indicate that residues in the helicase domain of nonstructural protein 3 can influence the protease supporting our hypothesis that full-length hepatitis C virus nonstructural protein 3 should be used for protease inhibitor optimization and .

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